Correlation between biochemical properties and adaptive diversity of skeletal muscle myofibrils and myosin of some air-breathing teleosts

Indian Journal of Biochemistry & Biophysics

Vol. 43, August 2006, pp. 217-225

Correlation between biochemical properties and adaptive diversity of skeletal

muscle myofibrils and myosin of some air-breathing teleosts

Riaz Ahmad and Absar-ul Hasnain*

Laboratory of Biochemical Genetics, Department of Zoology, Aligarh Muslim University, Aligarh 202 002, India

Received 3 November 2005; revised 22 June 2006

Functional properties of myofibrils and relative stability of myosin of five teleosts Channa punctata, Clarias

batrachus, Mastacembalus armatus, Labeo rohita and Catla catla adapted to different breathing modes were compared.

Myofibrillar contractility and m-ATPase of air-breathing organ (ABO) possessing C. punctata and C. batrachus were low

and least affected by pH in the range of 7.1-8.5. However, their myosin isoforms were relatively thermostable, more soluble

at sub-neutral pH values, between 0.1 to 0.15 M KCl concentrations and less susceptible to α-chymotryptic digestion. In

contrast, myofibrils and myosin of water-breather major carps L. rohita and C. catla were more contractile and susceptible

to pH and salt concentrations. Thus, correlation between catalytic efficiency and relative stability of myofibrils and myosin

of ABO-possessing teleosts was of reverse order and magnitude, as compared to water-breathers. Interestingly, myofibrils

and myosin of the behavioral air-breather M. armatus showed intermediate properties. The specific levels of m-ATPase of

all the five teleosts were in conformity with the levels of metabolic marker, the lactate dehydrogenase. The effect of

chymotryptic cleavage of 94 and 173 kDa domains on ATPase, individuality of peptide maps of MyHC isomers and

perturbation of phenylalanine residues by urea implicated hydrophobic residues in stabilizing myosin structure in these fish.

The present study suggests two apparent evolutionary modifications of myofibrils and myosin in ABO-possessing teleosts:

(i), ‘down-regulation’ of ATPase that explains sluggishness of such species and, (ii), more stable molecular structure to

support stress of air-breathing modes of life.

Keywords: Air-breathing teleosts, Chymotryptic Peptide maps, Difference spectra, Hydrophobic interactions, m-ATPase,

Muscle-type specificity, MyHC isoforms/isomers, SDS-PAGE, Structural plasticity

Temperature is widely regarded as evolutionary

affecter of the phenotypic plasticity of fish myosin1-3

,

which is the major contractile protein of myofibrils. A

few reports on urea tolerance of actomyosin ATPase

and myosin of elasmobranchs have also been

published4-6

. Urea has been reported to modify actin-

myosin interaction in a way that is different from

freshwater teleosts6. Exposure of thiols and confor-

mational stability of myosin of two elasmobranchs in

urea solutions has also been demonstrated5,6

.

However, reports about the influence of air-breathing

on specificity of contractile proteins of fish muscle

are lacking. Studies on LDH isozymes, however,

suggest that air-breathing teleosts preferentially utilize

anaerobic pathway to meet their energy needs during

hypoxia7,8

. The switchover to anaerobic metabolism

indicates adaptability of glycolytic pathway in the

muscle sarcoplasm. It is, therefore, conceivable that

contractile proteins of air-breathing teleosts have

undergone corresponding adaptive changes.

Myosin of air-breathing fishes should have ability

to function at elevated temperatures during land

excursions and survivability under mild desiccation

and pH changes. Phenotypic changes in fish myosin

result from the switchover to expression of alternative

heavy chain (MyHC) isoforms9-11

. The switchover

may occur during gross anatomical changes such as

development12-14

or during short duration acclimation

to different temperatures3,15,16

. ATP splitting (ATPase),

actin-binding and thick filament formability, all three

_______________

*Corresponding author

E-mail: [email protected]

Tel: +919358218737

Fax: +915712702885

Abbreviations: ABO, air-breathing organ; BPB, bromophenol

blue; DEAE, diethyl amino ethyl; LDH, lactate dehydrogenase;

m-ATPase, myofibrillar Mg2+-ATPase; Mf, myofibrils; MyHCs,

myosin heavy chains; MyLCs, myosin light chains; NAM, natural

actomyosin; PMSF, phenyl methyl sulfonyl fluoride; SDS-PAGE,

sodium dodecyl sulfate polyacrylamide gel electrophoresis;

TLCK, N-tosyl-L-lysine chloromethyl ketone; Tris, Tris

(hydroxymethyl) aminomethane; UV-DS, ultraviolet difference

spectra.

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006

218

essential activities of myosin are the functions of

MyHC. While ATPase and actin-binding sites are

located in twin globular heads of 400 kDa MyHC

dimer, its α-helical rod portion determines

solubility17,18

.

In the present study, an attempt has been made to

investigate the extent to which air-breathing has

modified myofibrillar contractility, m-ATPase,

myosin ATPase and structural plasticity of myosin

isomers. Since air-breathing was repeated several

times during evolution among quite distant groups of

fishes19

, we have selected teleosts which have air-

breathing organs (ABO) of different types and

origins. ABO-equipped species were: murrel, Channa

punctata and the catfish Clarias batrachus, while

Mastacembalus armatus, a behavioral air-breather has

no ABO and instead uses skin as accessory

respiratory organ19,20

. Major carps Labeo rohita and

Catla catla were taken as water-breather controls.

Materials and Methods

Acrylamide, ATP, bis-acrylamide, coomassie

brilliant blue R-250, DEAE-cellulose, PMSF, TLCK-

treated α-chymotrypsin and Tris (hydroxymethyl)

aminomethane were from Sigma Chemicals, USA and

2-mercaptoethanol was from CDH Chemicals, India.

Buffers and other salts used in the experiments were

of analytical grade purchased from Merck, CDH or

Qualigens, India.

Collection of fish

Fish murrel Channa punctata, Mastacembalus

armatus, the catfish Clarias batrachus and major carps

Labeo rohita and Catla catla were collected from

upper Ganges canal at Nanau, 20 km from Aligarh

during November, when water temperature at the site

was 12-15oC. Fish were transported to laboratory under

oxygen in polythene bags partly filled with water.

ABO-bearing species could be transported without this

arrangement. Fish were acclimated for 24 h in separate

tanks of 60 L capacity in the water of 15±2oC with

continuous aeration by bubbling. The density was

10 each for C. punctata and C. batrachus, 6 for

M. armatus and 4 each for the major carps.

Preparation of myofibrils or natural actomyosin (NAM) and

measurement of contractility

Fish were stunned by a severe blow to the head and

white muscle from anterior myotomes was carefully

removed, avoiding contamination of dark muscle.

Muscle pieces were immediately transferred to

polythene bags, which were dipped in ice bath to stop

glycolysis and delay rigor mortis. Myofibrils were

prepared as described earlier21

.

Natural actomyosin (NAM) from myofibrils was

prepared by extracting in 0.45 M KCl containing

25 mM phosphate buffer (pH 7.0), followed by preci-

pitation with 10 vol of distilled water22

. Precipitation

cycle was repeated three times and resulting preci-

pitate dissolved in 0.6 M KCl or NaCl containing

Tris-HCl buffer, pH 7.5.

The contractility was measured by the procedure

described previously21

with the modifications that pH

was a variable. It was estimated in 0.1 M KCl and

40 mM buffer at a specific pH and was defined as

(Pv0-PvATP)/Pv0×100, where Pv0 was the initial packed

volume of myofibrils and PvATP after adding 1 mM

Mg-ATP. Mfs were packed by centrifugation at

1000 rpm and 20°C for 10 min.

Lactate dehydrogenase (LDH) assay

First wash saved during preparation of myofibrils

was dialyzed against three changes of 50 mM Tris-

HCl. Precipitated material was removed as pellet,

following centrifugation at 10 K rpm and 4oC.

Supernatant was used for the assay of LDH activity.

Essentially, the protocol of Bergmeyer23

was

followed, while L-lactate was used as the substrate.

Assays were made in 60 mM phosphate buffer of pH

7.2, 1 mM NAD or 0.2 mM NADH at 20°C against

50 mM substrates.

Preparation of myosin and MyHC isomers

Myosin extraction was performed according to the

protocol24

, with the modification that prior to

extraction myofibrils were washed twice with 5 mM

MgCl2-ATP. The final pellet was suspended in

phosphate buffer (pH 8.0) to give a final concen-

tration of 0.05 M. This was followed by the addition

of MgCl2 and ATP to the final concentrations of

0.1 M and 0.005 M, respectively. The method gave a

fairly good yield (up to 250 mg/50 g muscle), with

only minor contamination of actin, which could be

removed by treating with DEAE-cellulose. Inclusion

of 0.001 M PMSF at each extraction step prevented

the frequent cleavage of MyHC due to intrinsic

proteases.

MyHC isomers from myosin were prepared

essentially as described previously25

. Briefly, myosin

was gently stirred in 2.4 M LiCl overnight. Tri-

potassium citrate was slowly added with continuous

and gentle stirring to a final concentration of 0.8 M.

AHMAD & HASNAIN: BIOCHEMICAL DIVERSITY OF MUSCLE PROTEINS OF AIR-BREATHING TELEOSTS

219

The thick and sticky precipitate, thus formed was

collected by centrifugation. After extensive dialysis

against 0.5 N NaCl in Tris-HCl (pH 7.5), followed by

centrifugation at 10 K rpm, the supernatant was taken

as MyHC preparation. It was screened by SDS-

PAGE and checked for Ca2+

-ATPase activity.

Concentration of myofibrils or myosin was deter-

mined by biuret method26

. Native myosin was also

read directly at 280/260 nm using the formula: Protein

concentration (mg/ml) = A280 × 1.5-A260 × 0.75.

ATPase assay

ATPase was assayed at 20°C and the liberated Pi

was colorimetrically determined22

. Assays were made

at 20°C, 0.05 M KCl, 1 mM MgCl2/CaCl2, 1 mM ATP

and 0.3 mg /ml protein concentration. Buffers were 25

mM Tris-maleate for pH 6.0 to 7.0 and Tris-HCl for

pH 7.5 to 8.5.

SDS-PAGE and molecular weight estimation

SDS-PAGE of Laemmli27

was employed to screen

the NAM, myosin or MyHC preparations. Gels con-

tained 10% acrylamide (A to C ratio 30:0.8), 1% SDS

and 10% glycerol. Dimensions of the gel slabs were

100 × 150 × 1 mm. Lower gels were made in 0.375 M

Tris-HCl (pH, 8.6) and upper gel (3%) in 0.125 M

Tris-HCl (pH, 6.8). Runs were made in Tris-glycine

of pH 8.3 (25 mM and 0.25 M, respectively). Routine

protein staining was performed with Coomassie

brilliant blue R-250. Electrophoretic patterns were

documented by digital imaging. Molecular weights

(Mr) of individual polypeptides were estimated by

GelPro software (Cybernetics, USA). Chicken NAM

was used as the molecular weight marker.

Purification of individual isoforms by preparative SDS-PAGE

Individual preparations of myosin from white

muscle of selected fish species were screened by the

preparative SDS-PAGE. MyHC bands were visua-

lized following 2 h washing in distilled water and then

placing the gels in cold. Gels were then washed with

distilled water and placed on ice over a polythene

sheet to visualize MyHC-SDS complex as white

bands. The bands were cut out and treated as

described earlier28

. Individual gel pieces with MyHC

were crushed and squeezed by smooth glass rods in

dialysis tubing in the presence of 1× running buffer

containing SDS. Electro-elution was carried out for

3 h and the contents were centrifuged at 10,000 g for

15 min. The supernatant containing a specific MyHC

isoform was saved and used for peptide mapping.

Electrophoresis of MyHC isoforms

MyHC isoforms were resolved by SDS-PAGE

essentially according to previously described proto-

col29

, with the modification that gels were 10% in

glycerol. Vertical slab gels (100 × 150 × 1 mm) con-

taining 8% acrylamide concentration (A:C= 200:1) in

the separating and 4% in stacking gel (A:C = 20:1)

were used. With the exception of lower-gel buffer that

was 0.75 M and pH of 9.3, other solutions and buffers

were the same

as in Laemmli’s protocol27

. Scion

imaging software programme was used for densito-

metry of individual MyHC isoform.

For urea-PAGE, molarity of Tris-HCl buffer in gel

and Tris-glycine in running buffer was the same as in

Laemmli’s27

protocol, except that SDS was replaced

by 5 M urea (final concentration) and no upper gel

was cast.

pH Dependence of solubility

Solubility of different myosins as a function of

variations in pH values and salt concentrations was

estimated as follows. Myosin solution (2.5 mg/ml)

was incubated with the buffers of variable salt

concentrations and desired pH for 3 h at ice

temperature. Buffers of different pH values were the

same as used for ATPase activity.

Students’ t-test was applied to check the significant

rise in solubility values (P<0.05). The values were the

average of duplicate determinations of three different

samples.

Kinetics of thermal inactivation of Ca2+-ATPase

Myosin (1.0-1.5 mg) in 0.5 M KCl containing 25

mM Tris-HCl of pH 7.5 was incubated at 45°C for

predetermined time intervals and the inactivation was

stopped by transferring the tubes to crushed ice.

Aliquots were also saved separately in the crushed ice

for turbidity measurement and read at 340 nm on

GENESYS-10 UV-Visible Spectrophotometer. Rate

constants of inactivation (kD) were calculated by the

formula = (ln Co- ln Ct)1/t, where Co and Ct were the

ATPase activities, before and after incubation time

t (in sec).

Limited proteolytic cleavage and changes in ATPase

It was monitored, by digesting white myotomal

muscle NAM of each species with TLCK-treated

α-chymotrypsin at 20°C and a ratio of 50:1. At

different time intervals, 1 ml aliquots were pipetted

out and 0.5 mM PMSF (final concentration) was

added to stop the proteolysis. Part of aliquots was

used for Ca2+

-ATPase assay22

or SDS-PAGE analysis27

.


220

Peptide mapping

MyHCs isolated from white myotomal muscle

were further purified electrophoretically. Initially,

SDS-PAGE in 10% gels

was performed27

. After

washing with distilled water, gels were placed on ice

over a polythene sheet. MyHC-SDS complex could be

visualized as white bands, which were cut out and

treated as described earlier28

. TLCK-treated α-chymo-

trypsin solution at a substrate to enzyme ratio=25:1

was added to each well. Electrophoresis was initiated,

but interrupted after 30 min and “within-gel diges-

tion” of MyHC was allowed to proceed for 1 h.

Electrophoresis was then resumed and continued till

the BPB line reached the end of the gel. Protein bands

were visualized by silver staining.

UV-difference (UV-DS) spectroscopy

For spectral analysis, solutions of 2 mg myosin/ml

(dissolved in 0.5 M KCl in 20 mM Tris-maleate of

pH, 7.0) were mixed with equal volume of 2x urea

solution of a specific strength5. All urea solutions

contained the myosin dissolution buffer (0.5 M KCl in

20 mM Tris-maleate of pH 7.0). UV-DS were

recorded after 2 h incubation at room temperature on

UV-5704 SS spectrophotometer (ECL, India).

Results

Concordance between pH dependence of contractility and

LDH level of muscle

Fig. 1 displays two important characteristics of

myofibrillar proteins: (i), at pH values of 7.0 to 8.0,

contractility of C. punctata and C. batrachus myofi-

brils was lower than of carps, and (ii), concordance of

specific myosin ATPase (Fig. 1B) with specific LDH

activities of muscle of corresponding species (Fig. 1C).

LDH is an established glycolytic marker of muscle

sarcoplasm. Profiles of m-ATPase are not shown here,

since for each species they were identical to contrac-

tility. Interestingly, contractility of M. armatus myo-

fibrils and LDH levels were intermediate to ABO-

possessing species and the carps.

Species-specificity of MyHC isoforms

MyHC prepared from white trunk muscle accor-

ding to Lied and Decken25

had no ATPase activity.

Also, no appreciable inter-species differences were

observed in the stacking of MyHC in routine SDS-

PAGE27

(Fig. 2A). MyHC preparations further puri-

fied by preparative SDS-PAGE were homogenous in

urea gels (Fig. 2B). Highly purified MyHCs of white

muscle of each of the five teleosts stacked as single

(monomorphic) bands in SDS-PAGE system of

Giullian et al.29

(Fig. 2C). The densitograms (Fig. 2D)

supported the inter-species variability in electro-

phoretic stacking of MyHC isomers displayed in

Fig. 2C.

KCl concentration and pH dependence of solubility profiles

Between ionic strength of 0.1 to 0.15 M (Fig. 3A),

myosin of ABO-possessing teleosts was 20-25% more

soluble than the homologues from major carps. The

maximum solubility at 0.2 M KCl was a common

property of all myosin isoforms. But, myosin of

ABO-possessing teleosts was distinct in displaying

10% higher solubility (P<0.05) between pH values of

6.5 and 7.1 (Fig. 3B).

Fig. 1—(A): pH Dependence of contractility of myofibrils from white myotomal muscle of teleosts [Experimental details are given under

Materials and Methods]. Cp, Channa punctata; Cb, Clarius batrachus; Ma, Mastacembelus armatus; Lr, Labeo rohita and Cc, Catla

catla; (B): pH Dependence of Mg2+-ATPase activity of white muscle myosin isoforms; and (C): Specific LDH activities in various white

muscle extracts [Values are expressed as mean ± SD of triplicate determinations of five individuals]


221

Effect of thermal incubation

Rate constants of inactivation kD calculated from

the first order plots of inactivation of myosin Ca2+

-

ATPase (Fig. 4A) were 5.74 × 10-4

s-1

and 5.85 × 10-4

s-1

for C. batrachus and C. punctata, respectively. The

kD values for myosin Ca2+

-ATPase of L. rohita, C.

catla and M. armatus were 20.18 × 10-4

s-1

, 19.93 ×

10-4

s-1

and 10.7 × 10-4

s-1

, respectively. Clear species

variations were displayed by the turbidity profiles also

(Fig. 4B); turbidity reached maxima at 20 min of

incubation at 45oC. Absorbance of carp myosin at this

peak time of denaturation was more than twice as

high as those of C. batrachus and C. punctata.

Turbidity profiles of myosin of M. armatus were in

between those of the myosin of major carps and of

ABO-possessing teleosts.

α-Chymotryptic cleavage of MyHC and its correlation with

ATPase

Fig. 5 compares proteolytic susceptibility at NAM

to chymotrypsin ratio of 50:1. SDS-PAGE profiles

demonstrated that it was essentially the cleavage of

200 kDa MyHC into low Mr polypeptides (Fig. 5A-

D). Myofibrillar proteins of C. punctata and C.

Fig. 2—(A): SDS-PAGE profiles of purified MyHC isoforms

isolated from white skeletal muscles of the teleosts [Abbreviations

of species name the same as in Fig. 1. The 10-13 µg protein was

loaded on to wells. Experimental details are given under

‘Materials and Methods’]; (B): PAGE patterns of purified MyHC

isoforms in 5 M urea [Order and loading was the same as in Fig.

2A]; (C): SDS-PAGE profiles of purified MyHC isoforms in the

system of Giulian et al.34 [chicken MyHC was used as the

marker]; and (D): Densitogram of SDS-PAGE profiles of purified

MyHC isoforms displayed in Fig. 2C, showing differences in

relative mobilities

Fig. 3—(A, B): Solubility of different myosins as a function of

salt concentrations and different pH values [Points in the plots are

average of duplicate determinations of three different

preparations. The plotted values were significant at P<0.05]

Fig. 4—(A, B): -log Plots of first order rate constants of

inactivation of Ca2+-ATPase activity at 45oC; and changes in

turbidity profiles of myosin during incubation at 45oC [Experi-

mental details of each of the above experiments are given in

‘Materials and Methods’]


222

batrachus were most stable. Following 10 min of

digestion, a fraction of undigested MyHC isomers

was retained as a thin band of 200 kDa in SDS-PAGE

profiles of these species (Fig. 5A-B). Fast degradation

of L. rohita and C. catla MyHC was concomitant with

the gradual increase in intensity of 173 kDa HMM-

HC and 94 kDa S1-HC bands (Fig. 5C-D). As

apparent from thickening of actin band, degradation

products co-stack with actin. Susceptibility of Ca2+

-

ATPase of these species was reflected by two main

characteristics (Fig. 5E-F). Either the magnitude of

activation was low as in carps (130-140%) or the

rapid decline followed as in M. armatus and L. rohita.

In contrast, Ca2+

-ATPase activation of 160-170%

occurred in NAM of C. punctata and C. batrachus

(Fig. 4E). In these species, activity was maintained at

100%, even after 80 min of digestion.

Sub-molecular diversity of peptide maps of MyHC isoforms

Substructural basis of differences between MyHC

isoforms of white skeletal muscle were compared by

peptide mapping in 15% gels, following digestion

with TLCK-treated α-chymotrypsin28

(Fig. 6). The

maps of MyHC of C. punctata, C. batrachus and M.

armatus consisted of 44, 35 and 32 polypeptides

respectively and 41 each for L. rohita and C. catla.

Conformational variations of myosins in urea solutions

Typical conformational changes at the lowest

(0.5 M) and highest molarity (4.0 M) of urea are

shown in Fig. 6A and B, respectively. With the

exception of L. rohita, each myosin showed a distinct

peak at ~265 and troughs at 259, 263 and 270 nm

(Fig. 7A-B). The wavelengths were essentially the

perturbation range of phenylalanine residues (Fig. 7A-

B). As buried phenylalanine residues got exposed, due

to increasing concentrations of urea, slight blue shift

in spectra also occurred, accompanied by the

appearance of new peaks. The ∆A values of deepest

trough at 268-270 nm were used to compare the

conformational differences as the function of molarity

of urea (Fig. 7C). It was obvious that ∆A values for

myosins of two ABO-possessing teleosts were

substantially lower than those of M. armatus and the

major carp myosin isoforms.

Fig. 5—SDS-PAGE profiles showing changes in sub-molecular

composition of polypeptides due to α-chymotryptic digestion of

NAMs at the substrate to protease ratio of 50:1 for 30 min. The

panels are (A): C. punctata; (B): C. batrachus; (C): L. rohita; and

(D): C. catla. Profiles of M. armatus (not included here) were

quite similar to those of major carps. Undigested chicken NAM is

used as marker. Changes in Ca2+-ATPase activity during course of

digestion: (E): NAMs of two ABO-possessing teleosts and M.

armatus (F): NAMs of the carps. Experimental details are given

under ‘Materials and Methods’.

Fig. 6—Peptide mapping of MyHC isomers from white skeletal

muscle of the teleosts obtained following α-chymotryptic

digestion [Lane M, undigested chicken actomyosin; and lanes 1-5,

C. punctata, C. batrachus, M. armatus, L. rohita and C. catla,

respectively. A total of 44, 35, 32, 41 and 41 peptides were

present in C. punctata, C. batrachus, M. armatus, L. rohita and C.

catla, respectively]


223

Discussion Partitioning into aerial and aquatic modes of

breathing is characteristic of ABO-possessing teleosts

Clarias batrachus and Channa punctata19,20

. As a

result, these species are capable of surviving outside

water and bypass water scarcity by aestivating in

mud. The sustenance outside water, however, depends

on the type of ABO. Air-breathing organs of

C. batrachus and C. punctata differ in anatomy,

organization, evolutionary origin and complexity20

.

M. armatus lacks any specialized ABO, but behaves

as an air-breather. Gaseous exchange takes place

through skin, which has a rich subcutaneous blood

supply19

. The two carps respire by aquatic mode only.

Thus, the investigated teleosts had different life styles

and modes of breathing. Biochemical evidence

presented here discriminate these fish broadly as air-

and water-breathers and also at species level.

For each species, plots of myofibrillar contractility

and m-ATPase were identical (Fig. 1A) and hence

independent of breathing mode. However, magnitude

of these properties was related with the mode of

breathing. ABO-possessing teleosts had distinctly

lower contractility and m-ATPase than the water-

breathing carps. The equivalence of these functions

with specific Mg2+

-ATPase of myosin suggests that

catalytic properties of myosin are of prime importance

for teleosts of either mode of life. The agreement

between specific m-ATPase of each species with the

activity levels of LDH (Fig. 1C) points to an

integrated evolution of glycolytic metabolism and

muscle motility.

MyHC, the core functional polypeptide assembly

displayed inter-species variations in electrophoretic

mobility of the isomers, suggesting structural

differences (Fig. 2). MyHC isomers of each species

were monomorphic, indicating that white muscle was

composed of single type of muscle fibers11

. Therefore,

the observed biochemical differences in myosin of

air- or water-breathing species cannot be attributed to

complexity of muscle fiber types. Mosaic muscles

composed of more than one type of muscle fibers

display the presence of several MyHC isomers9,10

.

Occurrence of monomorphic MyHC is consistent with

histochemical evidence on C. punctata and M.

puncalus, a sister species of M. armatus30

.

Ionic strength and pH dependence of solubility of

native myosin also revealed species differences (Fig.

3A-B). The solubility of stable myosin of ABO-

possessing teleosts between 0.1-0.15 M KCl was

higher, than that of unstable isomers of L. rohita and

C. catla, which obviously should be attributed to air-

breathing linked stability. Two arguments supported

this inference. First, due to less denaturation below

pH 7.0 (Fig. 4B) myosin isomers of ABO-possessing

teleosts should remain in a soluble state around 0.1 to

0.15 M (Fig. 3A). Second, solubility profiles of

myosin of even behavioral air-breather M. armatus

resembled closely with those of ABO-possessing

teleost isomers.

Thermal incubation revealed functional as well as

structural differences of the myosin isomers. Thermal

inactivation rate constants (kD) of myosin ATPase

were about four times as low as those of the major

Fig. 7—Typical UV-DS of unfolding profiles of various myosin isomers in urea solutions of (A): 0.5 M; and (B): 4.0 M final molanity,

respectively [The 2 mg myosin/ml (dissolved in 0.5 M KCl in 20 mM Tris-maleate, pH 7.0) was mixed with equal volume of 2x urea

solution of a specific strength containing myosin dissolution buffer (0.5 M KCl in 20 mM Tris-maleate of pH, 7.0). UV-DS were recorded

after 2 h incubation at room temperature]; and (C): Changes in the main trough in UV-DS at 268-270 nm, displaying effect of increasing

concentration of urea in various myosins [The spectra were average of duplicate determinations of three individual preparations.

Experimental details are same as given above]


224

carps. Thus, characteristics of ABO-possessing teleosts

myosin were low physiological (Mg2+

) ATPase and

substantially thermostable Ca2+

-ATPase (Fig. 4A).

The kD of ATPase inactivation for Cyprinus carpio

was reported to differ by a magnitude of 10 for a

temperature difference of 20oC

3. However, all of the

teleosts, in the present study, inhabited the water of

12-15oC, hence the differences in thermostability

were not due to acclimation to different temperatures.

The other parameter, turbidity also supported higher

thermostability of ABO-possessing teleosts. Turbidity

is a solubility related change and an indicator of

ability of inter-molecular aggregation. In case of fish

myosin, aggregation occurs by the interaction of

rod31

, rather than the head-to-head reaction typical of

mammalian myosin32

. Thermal denaturation was

carried out at a pH 7.5, where according to pH

dependence of the solubility, each of the native

myosin isoform was totally soluble (Fig. 4B).

Therefore, differences in turbidity profiles were due

to inter-species differences in aggregation behavior18

,

which in turn, reflect structural differences of myosin

isomers.

To compare susceptibility of ATPase domains,

chymotryptic cleavage of MyHC by SDS-PAGE and

ATPase assay were carried out simultaneously.

Cleavage of two domains of 173 kDa HMM-HC and

94 kDa S1 was parallel to decline in Ca2+

-ATPase

(Fig. 5). Since, we used TLCK-treated chymotrypsin,

it was free of trypsin contamination that would

generate HMM, due to different cleavage specificity.

MyHC of ABO-possessing teleosts was least

susceptible, since release as well as subsequent

cleavage of HMM-HC (Heavy Meromyosin Heavy

Chain) and S1 (subfragment-1) proceeded slowly.

Moreover, in these two species, a fraction of

undigested MyHC persisted in SDS-PAGE profiles

(Fig. 5A-B; lanes 2-3) up to 10 min of proteolysis. A

compact molecular structure or the absence of one or

more critical sites might account to the decreased

susceptibility. Either of the possibilities supports

differences in primary structure of MyHC isoforms.

Primary sequence of Cyprinus carpio myosin

provides an example of critical location of a specific

residue. Just a single lysine at position 301 in 50 kDa

domain (accession nos GenBank U8992 and U32574)

makes its MyHC highly susceptible to tryptic

cleavage24

. Chymotrypsin cleaves a polypeptide at

carboxyl groups contributed by phenylalanine,

tryptophan and tyrosine residues. Therefore, most

probably some of these hydrophobic residues had a

critical location in vicinity or within catalytic domains

of susceptible MyHC isomers.

More extensive variations in hydrophobic residues

were indicated by the peptide maps (Fig. 6). The

number of countable polypeptides in the maps varied

between 32-41, pointing to deletion/addition of a

number of specific sites. During evolution, a specific

protease cleavage site could be eliminated or added

by alteration in the reading frame of the gene due to

deletion or addition of a nucleotide. Hence, the

observed sub-molecular variations of MyHC isoforms

reflected their different evolutionary histories.

Few data are available on spectral comparisons of

fish myosin. Mirror carp myosin is known to acquire

random structure at rather low concentration (4.0 M)

of urea, while tyrosine residues of elasmobranch

myosin are not exposed at even 6.5 M urea5. In the

present study, no perturbation of tyrosine or

tryptophan was observed up to 4.0 M urea, for any of

the myosins. The exposure of buried phenylalanine

residues, however, occurred, which was also

accompanied by a blue shift as the molarity of urea

increased (Fig. 7A and B). The increase in ∆A at 268-

270 nm (Fig. 7C) displayed wide variations in the

magnitude of perturbation by urea. As obvious,

myosin isomers of ABO-possessing teleosts were

identifiable as one stable class against that of the

aquatic breathers. The chymotryptic cleavage and

UV-DS data suggested that hydrophobic interactions

substantially contributed to structural stabilization of

investigated myosin isoforms.

This is the first report that demonstrats modifi-

cation of ATPase, contractility and thermostability of

fish myofibrillar proteins, in context to breathing

modes. The data highlight two main adaptive charac-

teristics of myosin isomers of ABO-possessing

teleosts: (i) a ‘down-regulation’ of catalytic activity to

make muscle sluggish; and (ii) the exceptional stability

of myosin isomers to sustain stressful conditions. In

contrast, during low temperature acclimation,

catalytic efficiency (Mg2+

-ATPase) of teleost myosin

was enhanced while thermostability decreased3,33,34

.

Acknowledgements

The authors thankfully acknowledge the financial

grant from UGC (to AH) and to the Chairman,

Department of Zoology, for providing necessary

laboratory facilities. We also thank Prof. T M Haqqi

of Case Western Reserve University (USA), Prof.

Javed Musarrat of the Institute of Agriculture, Aligarh


225

Muslim University and Mr F R Khan, who helped us

in many ways.

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Documents

Correlation between biochemical properties and adaptive diversity of skeletal muscle myofibrils and myosin of some air-breathing teleosts